1. Field of the Invention
This invention relates generally to a competitive chemiluminescent assay for the detection of the presence of an analyte in a sample. More specifically, the present invention relates to a competitive assay for determining the concentration of an analyte (e.g., a cyclic nucleotide monophosphate such as cAMP) in a sample comprising cells wherein the cells are grown on an antibody coated plate.
2. Discussion of the Background
A wide variety of metabolic responses are keyed to release of intracellular cyclic adenosine monophospate (cAMP). In many cases, these responses are mediated by cAMP-dependent protein kinase which, in the presence of elevated cAMP levels, triggers a wide variety of activating reactions. Among the best known metabolic responses keyed to cAMP are the conversion of glycogen to glucose in the liver, as well as a variety of activities keyed to the glycogen/glucose energy cycle. The principle hormone in this cycle that induces a rise in cAMP is epinephrine. There are, however, a wide variety of other hormones that will also trigger cAMP release, which in turn keys a metabolic response mediated by the kinase. These include adrenocorticotropic hormone (ACTH), follicle-stimulating hormone (FSH), luteinizing hormone (LH), thyroid-stimulating hormone (TSH), parathyroid hormone, vasopressin and prostaglandin I. Accordingly, it is clear that cAMP levels in specific tissues of mammals, including humans, may be key indicators of a variety of hormonal functions and interactions.
cAMP is only the best known of the cyclic nucleotides. In general, cyclic nucleotides appear as monophosphates. Guanosine monophosphate (cGMP), uridine monophosphate (cUMP) and cytidine monophosphate (cCMP) may all importantly bear on a wide variety of hormonal functions and intercellular interactions that may be desirably measured. cAMP is the most studied of these xe2x80x9cmessengerxe2x80x9d cyclic nucleotides.
Assays, including competitive ELISA assays, for cAMP are known. A widely reported assay is available from Assay Design and is a colorimetric assay. Other immunoassay products are available from Amersham Biosciences, (a scintillation proximity assay), Perkin-Elmer (FLASHPLATE(copyright), ALPHAScreen(trademark), fluorescent polarization) as well as IGEN (electrochemiluminescence). The Assay Design embodiment employs assay kits (also available under the trademark BIOMOL(trademark)) in a classic example of a competitive ELISA assay, in which the strength of the signal is inversely proportional to the concentration of the cyclic nucleotide present. The kit from Assay Design is for measurement of light absorption. A fluorescent assay kit is available from Molecular Devices.
Because of the very low values of cyclic nucleotides that may need to be detected in a variety of tissue samples, high sensitivity is often required. Many of the commercially available assays for cAMP itself do not offer this sensitivity and, therefore, require acetylation of the cAMP to promote better antibody binding for greater sensitivity. It would therefore be desirable to improve the sensitivity of cAMP assays. Such heightened sensitivity would be especially useful in assays for determining the effect of G proteins (e.g., GI and GS proteins) on cAMP levels. Such assays could be useful to help elucidate signal transduction mechanisms.
In addition, conventional assays are very expensive, due in part to the numerous steps necessary to complete them. Therefore, a need also exists for assays that require fewer steps and are therefore easier to automate and less expensive to run.
Accordingly, it remains a goal of those of skill in the art to find a competitive immunoassay that requires fewer steps and that is highly sensitive, offers a broad dynamic range, and employs reagents that can be obtained through simplified procedures.
According to a first aspect of the invention, a competitive immunoassay for detecting the amount of an analyte in a sample comprising cells is provided. The immunoassay includes steps of: providing an assay plate comprising one or more wells, wherein the wells are coated with a capture antibody; adding the sample to the one or more wells; growing the cells of the sample in the one or more wells; lysing the cells in the one or more wells; combining with the cell lysates in the one or more wells, (1) a conjugate of the analyte and an enzyme and (2) a primary antibody that is bound by the capture antibody and that binds, when so bound, the analyte to form a reaction mixture; incubating the reaction mixture to permit binding of the primary antibody and the conjugate; washing the reaction mixture to remove unbound conjugate or antibody; adding to the reaction mixture in the one or more wells a substrate comprising an enzyme labile group, wherein the enzyme of the conjugate is capable of cleaving the enzyme labile group of the substrate; and measuring a signal resulting from the cleavage of the enzyme labile group. The signal can be used to determine the presence and/or the concentration of the analyte in the sample. The analyte can be a cyclic nucleotide phosphate such as cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), cyclic uridine monophosphate (cUMP) or cyclic cytidine monophosphate (cCMP). The enzyme can be alkaline phosphatase. The substrate can be a chemiluminescent substrate, a fluorescent substrate or a calorimetric substrate. Preferably, the substrate is a chemiluminescent 1,2-dioxetane substrate.
According to a second aspect of the invention, a competitive immunoassay for detecting the amount of an analyte in a sample comprising cells is provided. The immunoassay includes steps of: providing an assay plate comprising one or more wells, wherein the wells are coated with a capture antibody; adding the sample to the one or more wells; growing the cells of the sample in the one or more wells; lysing the cells in the one or more wells; combining with the cell lysate in the one or more wells, (1) a tagged analyte and (2) a primary antibody that is bound by the capture antibody and that binds, when so bound, the analyte to form a reaction mixture; incubating the reaction mixture to permit binding of the primary antibody and the tagged analyte; washing the reaction mixture to remove unbound tagged analyte or antibody; and detecting a signal from the tagged analyte remaining in the one or more wells. The signal can be used to determine the presence and/or the concentration of the analyte in the sample. The analyte can be modified with an enzyme, or tagged with a fluorescent or a radioactive tag.